NIH-funded investigators are seeking support under the Shared Instrumentation Grant Program to obtain an Andor Revolution WD spinning disk confocal microscope. This unit is a platform for a broad-based system that combines high-speed spinning disk technology with novel excitation-scanning hyperspectral imaging (HIFEX). The resulting Andor/HIFEX system is completely novel and represents an enormous advance in performance and experimental versatility. It will provide vital high-speed multi-fluorophore 3-d imaging capability for a wide range of investigations involving live cells and tissues. The current application targets key research projects at the USA College of Medicine that critically need the cutting-edge capabilities of the Andor/HIFEX system. These high-priority projects include ongoing studies in the areas of Pulmonary Physiology and Pathology, Real-time Cardiovascular Signaling, Infectious Disease/Immunology and Cell Biology. Major users include Drs. Rich, Leavesley, Stevens, and Cioffi whose projects focus on crucial aspects of in situ signaling along the endothelial barrier of the lung. Their proposed studies will evaluate spatio- temporal aspects of crucial signals altering endothelial permeability in the lung and contributing to chronic and acute disease. Elucidating these complex signaling profiles at the cellular and tissue levels is extremely important to the mission of the Center for Lung Biology and to the NIH. Minor users include Drs. Fortwendel and Barrington who are addressing mechanisms of infection and immune response at levels not previously attainable through optical imaging. Additional minor users, Drs. Lin and Taylor, will examine the highly dynamic interactions of signals and effectors (such as Ca2+, nitric oxide and K+ channels) within the restricted space of the myoendothelial interface in intact blood vessels. Acquisition and implementation of the Andor/HIFEX system is justified: 1) Our three confocal microscopes at the University of South Alabama BioImaging Core Facility (BCF) - a Leica SP2, a PerkinElmer RS3 and a Nikon A1R - are heavily used and two of the units are quite old (14 and 9 years, respectively) and in increasing disrepair, and 2) the current imaging systems simply cannot provide key capabilities required by NIH-funded investigators. Critical equipment and personnel are in place, and we successfully assembled and tested this imaging approach in a recent equipment demonstration. In brief, implementing the Andor/HIFEX imaging unit will revolutionize our ability to perform simultaneous acquisition of multiple fluorescence signals in live cells and tissues, and will push ongoing NIH-funded research projects beyond current limitations. In the long range, including the Andor/HIFEX system within the BioImaging Core Facility at USA College of Medicine will advance existing projects and allow rapid pursuit of new experimental directions. This will be an invaluable tool for seeding collaborative and translational initiatives among our research faculty.